Rosalind B. Penney

Potential Role of UGT1A4 Promoter SNPs in Anastrozole Pharmacogenomics

Anastrozole belongs to the nonsteroidal triazole-derivative group of aromatase inhibitors. Recently, clinical trials demonstrated improved antitumoral efficacy and a favorable toxicity with third-generation aromatase inhibitors, compared with tamoxifen. Anastrozole is predominantly metabolized by phase I oxidation with the potential for further phase II glucuronidation. It also, however, is subject to direct N-glucuronidation by UDP-glucuronosyltransferase 1A4 (UGT1A4). Anastrozole pharmacokinetics vary widely among patients, but pharmacogenomic studies of patients treated with anastrozole are sparse. In this study, we examined individual variability in the glucuronidation of anastrozole and its association with UGT1A4 promoter and coding region polymorphisms. In vitro assays using liver microsomal preparations from individual subjects (n = 96) demonstrated 235-fold variability in anastrozole glucuronidation. Anastrozole glucuronidation was correlated (r = 0.99; P < 0.0001) with lamotrigine glucuronidation (a diagnostic substrate for UGT1A4) and with UGT1A4 mRNA expression levels in human liver microsomes (r = 0.99; P < 0.0001). Recombinant UGT1A4 catalyzed anastrozole glucuronidation, which was inhibited by hecogenin (IC50 = 15 µM), a UGT1A4 specific inhibitor. The promoter region of UGT1A4 is polymorphic, and compared with those homozygous for the common allele, lower enzymatic activity was observed in microsomes from individuals heterozygous for −163G<A, −219T<G, and −217C<T (P = 0.009, P = 0.014, and P = 0.009, respectively). These results indicate that variability in glucuronidation could contribute to response to anastrozole in the treatment of breast cancer.

Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines.

BACKGROUND Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Glutamine provides additional carbon and nitrogen sources for cell growth. The first step in glutamine utilization is its conversion to glutamate by glutaminase (GLS). Glutamate is a precursor for glutathione synthesis, and we investigated the hypothesis that glutamine drives glutathione synthesis and thereby contributes to cellular defense systems. METHODS The importance of glutamine for glutathione synthesis was studied in H460 and A549 lung cancer cell lines using glutamine-free medium and bis-2-(5-phenyl-acetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) a GLS inhibitor. Metabolic activities were determined by targeted mass spectrometry. RESULTS A significant correlation between glutamine consumption and glutathione excretion was demonstrated in H460 and A549 tumor cells. Culturing in the presence of [(13)C5]glutamine demonstrated that by 12h >50% of excreted glutathione was derived from glutamine. Culturing in glutamine-free medium or treatment with BPTES, a GLS-specific inhibitor, reduced cell proliferation and viability and abolished glutathione excretion. Treatment with glutathione-ester prevented BPTES-induced cytotoxicity. Inhibition of GLS markedly radiosensitized the lung tumor cell lines, suggesting an important role of glutamine-derived glutathione in determining radiation sensitivity. CONCLUSIONS We demonstrate here for the first time that a significant amount of extracellular glutathione is directly derived from glutamine. This finding adds yet another important function to the already known glutamine dependence of tumor cells and probably tumors as well. GENERAL SIGNIFICANCE Glutamine is essential for synthesis and excretion of glutathione to promote cell growth and viability.

A Review of the Field on Children’s Exposure to Environmental Contaminants: A Risk Assessment Approach

Background: Children must be recognized as a sensitive population based on having biological systems and organs in various stages of development. The processes of absorption, distribution, metabolism and elimination of environmental contaminants within a child’s body are considered less advanced than those of adults, making them more susceptible to disease outcomes following even small doses. Children’s unique activities of crawling and practicing increased hand-to-mouth ingestion also make them vulnerable to greater exposures by certain contaminants within specific environments. Approach: There is a need to review the field of children’s environmental exposures in order to understand trends and identify gaps in research, which may lead to better protection of this vulnerable and sensitive population. Therefore, explored here are previously published contemporary works in the broad area of children’s environmental exposures and potential impact on health from around the world. A discussion of children’s exposure to environmental contaminants is best organized under the last four steps of a risk assessment approach: hazard identification, dose-response assessment, exposure assessment (including children’s activity patterns) and risk characterization. We first consider the many exposure hazards that exist in the indoor and outdoor environments, and emerging contaminants of concern that may help guide the risk assessment process in identifying focus areas for children. A section on special diseases of concern is also included. Conclusions: The field of children’s exposures to environmental contaminants is broad. Although there are some well-studied areas offering much insight into children exposures, research is still needed to further our understanding of exposures to newer compounds, growing disease trends and the role of gene-environment interactions that modify adverse health outcomes. It is clear that behaviors of adults and children play a role in reducing or increasing a child’s exposure, where strategies to better communicate and implement risk modifying behaviors are needed, and can be more effective than implementing changes in the physical environment.

Lack of correlation between in silico projection of function and quantitative real-time PCR-determined gene expression levels in colon tissue

There are a number of in silico programs that use algorithms and external web sources to predict the effect of single nucleotide polymorphisms (SNPs). While many of these programs have been shown to predict accurately the effect of SNPs in functional areas of the gene, such as 5′ upstream or coding regions, empiric research may be warranted to confirm the functional consequences of SNPs that are predicted to have little to no effect. We compared predictions from FASTSNP (Function Analysis and Selection Tool for Single Nucleotide Polymorphism) and F-SNP (Functional Single Nucleotide Polymorphism) with experimentally derived genotype-phenotype correlations to determine the accuracy of these programs in predicting SNP functionality. We used normal colon tissue to evaluate 24 TagSNPs within six genes. Two of 16 SNPs that were predicted to have no functional effect in FASTSNP were significantly associated with gene expression. Only one of the eight SNPs that were predicted to have a low to high effect was significantly associated with gene expression. While the two in silico programs that were used were similar in their results for the SNPs predicted by FASTSNP to have no effect, of SNPs with scores from low to high, there were three that received an F-SNP score below what is considered functionally significant. In silico programs can fail to identify functional SNPs, supporting a continuing role for empiric analysis of SNP function. Laboratory analysis is necessary to identify causal SNPs accurately, establish biological plausibility of the effect, and ultimately inform cancer prevention strategies.

Effect of MRP2 and MRP3 Polymorphisms on Anastrozole Glucuronidation and MRP2 and MRP3 Gene Expression in Normal Liver Samples.

Anastrozole is an aromatase inhibitor (AI) used as adjuvant therapy for breast cancer. Anastrozole is subject to direct glucuronidation catalyzed by UDP-glucuronosyltransferase1A4 (UGT1A4). Interindividual variability in anastrozole glucuronidation may be affected by UGT1A4 SNPs. Interplay between drug metabolizing genes such as UGT1A4 and transporter genes may also be affected by genetic variability. Thus, we hypothesize that genetic variability in MRPs could influence anastrozole glucuronidation. The correlation between UGT1A4 and MRP2 or MRP3 transporter gene expressions and the correlation between MRP2 or MRP3 mRNA and anastrozole glucuronidation were analyzed in normal human liver samples. MRP2 and MRP3 mRNA levels were significantly correlated with UGT1A4 mRNA, with anastrozole glucuronidation and with each other (p<0.05). The data also demonstrated that MRP2 SNPs are positively correlated with MRP2 mRNA expression, while there was no association between MRP3 SNPs from this study and MRP3 expression. Significant correlations (p<0.05) between certain MRP2 SNPs (3972C>T, 2366C>T and -24C>T) and anastrozole glucuronidation were observed. There were no observed correlations between MRP3 SNPs and anastrozole glucuronidation. MRP2 polymorphisms have been identified as playing a role in the disposition of other drugs, and the data presented here indicate for the first time that MRP2 SNPs could influence anastrozole metabolism and contribute to interindividual variation in treatment responses.

Abstract 1298: The effect of adipocyte-derived factors on lung cells: Exploring the protective nature of excess weight on lung cancer risk

Lung cancer is the second most common cancer in men and in women (not including skin cancer) and the most common cause of cancer mortality. According to several epidemiologic papers, excess weight, identified as the BMI categories of overweight and obese, lowers lung cancer risk. Despite the numerous studies describing this anomaly, the underlying mechanisms for the protective effects are still unknown. Adipose tissue is known to secrete inflammatory molecules such as IL6, IL1β, and IL12. Activated IL6- and IL1β-associated inflammatory pathways are known to promote tumorigenesis, while the activated IL12-associated pathway inhibits tumorigenesis. We hypothesize that adipocytes create an environment that prevents lung carcinogenesis through activating inflammatory pathways that inhibit tumor growth and through blocking inflammatory pathways that promote tumor growth. To study the effect of adipocyte-derived factors on lung cells, alveolar fibroblasts (MRC5) and lung carcinoma (A549) cells were cultured in standard medium and in a 50:50 standard: adipocyte-conditioned medium (CM). In addition, cells were treated with benzo[a]pyrene (B[a]P), a known lung carcinogen. After 48 hours, mRNA expression levels of inflammatory genes were determined by RT-PCR. The data demonstrate that B[a]P treatment induced expression of IL1β, IL6 and IL12 in both cell lines. CM treatment decreased expression of IL1β and IL6 in MRC5 cells and increased their expression in A549 cells. In contrast, CM treatment decreased expression of IL12 in both cells lines. Cells treated with B[a]P plus CM had increased expression of IL12 in both cell lines, and reduced expression of IL1β and IL6 in MRC5 (2.3-fold and 6.5-fold, respectively) and A549 cells (40-fold and 11.5-fold, respectively) over B[a]P-treated cells. It was demonstrated that combined treatment increased NFκB (a transcription factor for IL1β, IL6 and IL12) mRNA expression 4-fold over control. The data suggest that conditioned medium inhibits B[a]P-induced and NFκB-mediated expression of IL1β and IL6 but increases NFκB-mediated expression IL12. This suggests that excreted factors from an adipocyte-rich environment may lead to the activation of growth-prohibiting inflammatory pathways, as opposed to growth-promoting pathways. Understanding these mechanisms and the possible discovery of biomarkers can be the foundation for new preventive techniques and treatments for lung cancer. Citation Format: Rosalind B. Penney, Daniel Sappington, Eric Siegel, Gunnar Boysen, Susan Kadlubar. The effect of adipocyte-derived factors on lung cells: Exploring the protective nature of excess weight on lung cancer risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1298. doi:10.1158/1538-7445.AM2015-1298

CYP19A1 single nucleotide polymorphism associations with CYP19A1, NFκB1, and IL6 gene expression in human normal colon and normal liver samples

Background Estrogen is known to decrease the risk of colon cancer in postmenopausal women, and may exert its actions by decreasing interleukin-6 (IL6) production via stabilization of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Estrogens are biosynthesized by CYP19A1 (aromatase), so it is possible that genetic variations in CYP19A1 influences the risk of colon cancer by altering expression of CYP19A1. Further, studies on gene-gene interactions suggest that single nucleotide polymorphisms in one gene may affect expression of other genes. The current study aims to explore the role of CYP19A1 single nucleotide polymorphisms on CYP19A1, NFκB1 and IL6 gene expression. Methods Phenotype–genotype associations, cross-associations between genes, and haplotype analyses were performed in both normal human colon (n=82) and liver (n=238) samples. Results CYP19A1 rs10459592, rs1961177, and rs6493497 were associated with CYP19A1 expression in colon samples (P=0.042, P=0.041, and P=0.013, respectively). CYP19A1 single nucleotide polymorphisms (rs12908960, rs730154, rs8025191, and rs17523880) were correlated with NFκB1 expression (P=0.047, P=0.04, P=0.05, and P=0.03, respectively), and CYP19A1 rs11856927, rs2470152, and rs2470144 (P=0.049, P=0.025, P=0.047, respectively) were associated with IL6 expression in the colon. While rs730154 and rs17523880 could not be analyzed in the liver samples, none of the other associations with the colon were replicated in the liver samples. Haplotype analysis revealed three separate haplotypes of the CYP19A1 single nucleotide polymorphism that were significantly associated with CYP19A1, NFκB1, and IL6 gene expression. Conclusion CYP19A1 single nucleotide polymorphisms are associated not only with CYP19A1 expression but also with NFκB1 and IL6 expression. These data demonstrate the possible functional consequences of genetic variation within the CYP19A1 gene on other genes in a biologically plausible pathway.

PARP1 Is Up-Regulated in Non-small Cell Lung Cancer Tissues in the Presence of the Cyanobacterial Toxin Microcystin

Non-small cell lung cancer (NSCLC) is the major form of lung cancer, with adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being its major subtypes. Smoking alone cannot completely explain the lung cancer etiology. We hypothesize that altered lung microbiome and chronic inflammatory insults in lung tissues contribute to carcinogenesis. Here we explore the microbiome composition of LUAD samples, compared to LUSC and normal samples. Extraction of microbiome DNA in formalin-fixed, paraffin-embedded (FFPE) lung tumor and normal adjacent tissues was meticulously performed. The 16S rRNA product from extracted microbiota was subjected to microbiome amplicon sequencing. To assess the contribution of the host genome, CD36 expression levels were analyzed then integrated with altered NSCLC subtype-specific microbe sequence data. Surprisingly phylum Cyanobacteria was consistently observed in LUAD samples. Across the NSCLC subtypes, differential abundance across four phyla (Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes) was identified based on the univariate analysis (p-value < 6.4e-4 to 3.2e-2). In silico metagenomic and pathway analyses show that presence of microcystin correlates with reduced CD36 and increased PARP1 levels. This was confirmed in microcystin challenged NSCLC (A427) cell lines and Cyanobacteria positive LUAD tissues. Controlling the influx of Cyanobacteria-like particles or microcystin and the inhibition of PARP1 can provide a potential targeted therapy and prevention of inflammation-associated lung carcinogenesis.

Diagnosis of lung tumor types based on metabolomic profiles in lymph node aspirates

BACKGROUND Treatment of lung cancer is evolving from the use of cytotoxic drugs to drugs that interrupt pathways specific to a malignancy. The field of metabolomics has promise with respect to identification of tumor-specific processes and therapeutic targets, but to date has yielded inconsistent data in patients with lung cancer. Lymph nodes are often aspirated in the process of evaluating lung cancer, as malignant cells in lymph nodes are used for diagnosis and staging. We hypothesized that fluids from lymph node aspirates contains tumor-specific metabolites and are a suitable source for defining the metabolomic phenotype of lung cancers. PATIENTS AND MATERIALS Metabolic profiles were generated from nodal aspirates of ten patients with adenocarcinoma, ten with squamous cell carcinoma, and ten with non-malignant conditions using time-of-flight mass spectrometry. In addition, concentrations of selected metabolites participating in the kynurenine and glutathione pathways were measured in a second set of aspirates using tandem mass spectrometry. RESULTS A list of consensus features that separated these three groups was identified. Two of the consensus features were tentatively identified as kynurenine and as oxidized glutathione. It was shown that metabolite concentrations in these pathways are different for patients with and without malignancy. CONCLUSION Together the data suggest that metabolomic analysis of lymph node aspirates can identify tumor-specific differences in cancer metabolism and reveal novel therapeutic targets. This proof-of-concept study demonstrates the validity to complement and refine diagnosis of lung cancer based on metabolic signature in lymph node aspirates. MICRO ABSTRACT Treatment of lung cancer is evolving from the use of cytotoxic drugs to drugs that interrupt metabolic pathways specific to a malignancy. We report here in that the metabolic phenotype of lung cancer can be determined in lymph node aspirates harboring malignant tumor cells. Knowledge about metabolic activity of malignant tumor cells may aide to personalize therapy.

Abstract 1041: Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines

Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Glutamine provides additional carbon and nitrogen sources for cell growth. The first step in glutamine utilization is its conversion to glutamate by glutaminase (GLS). Glutamate is a precursor for glutathione synthesis, and we investigated the hypothesis that glutamine drives glutathione synthesis and thereby contributes to cellular defense systems. The importance of glutamine for glutathione synthesis was studied in H460 and A549 lung cancer cell lines using glutamine-free medium and BPTES a GLS inhibitor. Metabolic activities were determined by targeted mass spectrometry. A significant correlation between glutamine consumption and glutathione excretion was demonstrated in H460 and A549 tumor cells. Culturing in the presence of [13C5]glutamine demonstrated that by 12 hrs >50% of excreted glutathione is derived from glutamine. Culturing in glutamine-free medium or treatment with Bis-2-(5-phenyl-acetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a glutaminase (GLS)-specific inhibitor, reduced cell proliferation and viability, and abolished glutathione excretion. Treatment with glutathione-ester prevented BPTES induced cytotoxicity. Inhibition of GLS markedly radiosensitized the lung tumor cell lines, suggesting an important role of glutamine-derived glutathione in determining radiation sensitivity. We demonstrate here for the first time that a significant amount of extracellular glutathione is directly derived from glutamine. This finding adds yet another important function to the already known glutamine dependence of tumor cells and probably tumors as well. Citation Format: Daniel Sappington, Rosalind Penney, Eric Siegel, Gunnar Boysen. Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1041.